Alkylated DNA base damage is cytotoxic and mutagenic unless repaired, and is prototypic of most DNA damage involving the chemical modification of DNA bases. DNA alkylation repair is therefore critical for genome stability and is furthermore a major resistance factor for cancer chemotherapies. DNA-glycosylases that remove alkylated bases by base-excision repair (BER) are relatively well characterized. However, two critical aspects of alkylation damage repair are still poorly understood: 1) the structural chemistries for alkylation damage reversal and 2) the crosstalk connecting alkylated base damage responses to other repair pathways. Overall, the two Specific Aims of this Project will address the challenge of characterizing at the molecular level the major alkylation damage reversal proteins in humans, and the critical crosstalk connecting alkylation damage to other repair pathways. The aims center on the human systems where possible and employ model organisms where needed, to reveal the core structural biochemistry of the well-conserved DNA repair machinery. We propose to characterize these poorly understood reversal and crosstalk repair mechanisms by integrating chemical, mutational, and biochemical approaches with two complementary structural techniques of macromolecular x-ray crystallography (MX) and small angle x-ray scattering (SAXS) in solution. Thus, this work will appropriately leverage and integrate the research accomplishments, strengths, and programs of the investigators and their institutions to promote, develop, and test a unified understanding of alkylation damage response proteins and novel inhibitors. Quantitative characterization of protein structures and complexes by MX and SAXS along with biophysical methods and computational analyses in the Tainer lab will be coordinated with detailed in vitro and in vivo biochemical and mutational results from the Pegg lab. In our analyses, we will address three fundamental hypotheses: 1) Damage specificity comes from the sum of multiple sub-steps that promote substrate recognition, which can be experimentally dissected and characterized. 2) Initial stages provide commitment to the multi-step repair pathways. These stages involve conformational changes forming stable DNA product complexes for handoffs, so sequential orchestration of repair steps is in part governed by binding affinity and interface exchanges. 3) Proteins can control crosstalk and connections with other repair pathways through DNA sculpting, to create recruitment platforms that promote DNA binding by proteins that initiate another distinct repair pathway. These ideas of summed specificity steps, protein-DNA product complexes for sequential handoffs, and protein-directed DNA sculpting to promote pathway connections have been elucidated from our results. These concepts therefore guide our current efforts to decipher the dynamic interplay of alkylated base reversal and repair proteins. Overall, these results will provide a unified understanding of alkylation damage responses relevant to characterizing their role in genetic integrity and resistance to chemotherapeutics, and to promoting advances in cancer therapies.